The type III secretion system of Gram-negative bacterial pathogens creates one of the most direct interfaces between pathogens and their hosts. These `needle-like' molecular machines inject bacterial effector proteins directly into host cells for the purpose of destroying an innate immune response and facilitating bacterial replication, dissemination, and disease progression. Effector proteins are unique virulence factors in that they often capture or mimic the properties of host signal transduction molecules. The present study focuses on three important bacterial type III effector families. First, we will interrogate a large family of bacterial Guanine-nucleotide exchange factors (GEFs) required for Salmonella, Shigella, and enterohaemorrhagic E. coli pathogenesis, respectively, through their common ability to activate Rho-family GTPase signaling cascades. The studies described here will advance recent high-throughput genetic screening approaches to identify bacterial effector protein localization within the host cellular environment. Findings from these preliminary studies will be applied to uncover the system dynamics of host-pathogen interactions responsible for Shigella invasion, and particularly the role of host acidic phospholipids on bacterial GEF signaling functions (Aim 1). Second, we will characterize the novel enzymatic mechanism of the Invasion plasmid antigen J (IpaJ) family of bacterial cysteine proteases that catalyze the proteolytic elimination of N-myristoyl modifications on host ARF GTPase cellular substrates. Insights gleaned from these studies will be advanced through a detailed analysis of Shigella innate immune pathway evasion, and specifically the role of protein demyristoylation in this process (Aim 2). Finally, we will investigate orphan members of the Shigella E3-ubiquitin ligase superfamily, and specifically their ability to modulate host immune components through protein ubiquitylation (Aim 3). Developing new drugs that target bacterial and host enzyme complexes would be an innovative approach to combat emerging antibiotic resistant microbes. Therefore, by revealing molecular details of type III effector family functions, from biochemistry to systems biology, we will uncover sites of potential weakness in bacterial pathogens that may be exploited for therapeutic intervention. Importantly, these studies will also provide new insights into the pathogenic mechanisms of numerous infectious disease agents and also into the biology of the human host.